Use of double-stranded RNA as a therapeutic, or to enhance RNAi-based reactions, are topics of interest within the health care and biotechnology industries. However, effective in vitro production of RNA has hindered these interests. In addition, inadequate in vitro production of RNA has limited advancement into other potential fields of RNA use.
Information transfer, and in particular, in vitro synthesis reactions, have traditionally focused on the path of genetic information transfer within a cell that stores information as dsDNA, i.e., from DNA to DNA, from DNA to RNA, and from RNA to protein, i.e., from DNA to RNA to protein. The study of RNA synthesis directly from an RNA template has traditionally received little academic or commercial effort, as few in vivo mechanisms for RNA to RNA transfer have been discovered.
Over the past ten to fifteen years, however, this trend has begun to change with the characterization of RNA viruses and their capacity for in vivo copying of single-stranded RNA to double-stranded RNA. (See, for example, Neufeld et al., 1991, J. Biol. Chem. 266(35) 24212-24219). Developments in viral-based genomics, as well as in studies of sequence-specific, RNA-triggered gene silencing, have provided a class of enzymes involved in the potential in vivo synthesis of RNA from RNA template. This class of enzymes, termed RNA-dependent or RNA-directed RNA polymerases (RdRps), are believed to be involved in just such RNA to RNA transfer roles.
It is believed that RdRps had a role in early evolution, when RNA was the primary genetic material of most, if not all, organisms, and used in the synthesis of RNA from RNA. RdRps have been identified in certain RNA viruses, e.g., polio and HIV, as well as in several known eukaryotes. RdRps are involved in genomic replication, mRNA synthesis, RNA recombination, and other like processes. Studies into RdRps have focused on characterization of these enzymes and their potential use in gene silencing studies as well as for targets in drug therapies directed at slowing down or eliminating a RNA viral load from a patient.
Use of RdRps as a tool to replicate an RNA template in vitro has thus far focused solely on non-exponentially forming a double-stranded RNA product from a single-stranded RNA template. RdRps that extend from secondary structure within the templates, as well as non-specifically, have been identified and are in the process of being characterized (U.S. patent Publication No. 2003/0124559 to Makeyev et al). No exponential in vitro amplification reactions have been devised to the inventor's knowledge, especially where oligonucleotide primers are involved. Rather, template ssRNA is replicated in a linear fashion.
Alternatively, it has recently been disclosed that anti-sense strands of RNA can be synthesized from a sense strand of RNA template, as long as DNA is utilized as an intermediate product of the reaction. This method, termed nucleic acid sequence based amplification (NASBA), calls for the reverse transcription of a sense strand of RNA into an antisense strand of DNA. Reverse transcription is conducted from RNA primed with synthetic oligonucleotides containing T7 RNA polymerase promoter sequence. The original strand of RNA is degraded with RNase H, leaving a single strand of DNA, which is converted back to an antisense strand of RNA using a primer to the DNA strand, and T7 RNA polymerase (Van Gelder et al., Proc. Natl. Acad. Sci., USA, 1990, 87(5):1663-1667; HIV QT Nov. 13, 2001). The NASBA method is problematic as RNA must be transcribed to DNA and converted back to RNA using two enzymes, a reverse transcriptase and a T7 polymerase (each having a different fidelity and processivity), as well as the design and preparation of primers having T7 RNA polymerase promoter sequence. These DNA intermediate based RNA amplification reactions also are not exponential in the amplification of product, thereby limiting their effectiveness.
Against this backdrop the present invention has been developed.